Carbohydrate-containing external invertase of Saccharomyces cerevesiae will be used as a model system for studying the biosynthesis of a secreted glycoprotein of known composition. Partially glycosylated invertase intermediates in the subcellular rough membrane, internal pool, and plasma membrane fractions will be isolated by immunoprecipitation from extracts of yeast protoplasts labeled with (3H)amino acids and (14C)glucosamine or mannose. Comparative compositional analyses of the recovered material in relation to the secreted holoenzyme should provide information on the sites of carbohydrate attachment and confirm whether initial sugar addition occurs by transfer to the peptide of a large lipid-linked oligosaccharide moiety as found in higher envaryotic systems. The kinetics of labeling of specific dolichol intermediates and the presence of sugar transferases in relation to invertase will be examined to clarify the pathway involved in this glycoprotein's synthesis. The role of glucose in the lipid-linked oligosaccharide transfer and its removal from newly glycosylated invertase will be assessed. Additional studies will determine what effect the glycosylation inhibitors, 2-deoxy-D-glucose and tunicamycin, have on the fate of the invertase peptide. It is planned to apply the results of these studies to the development of an in vitro system capable of the de novo synthesis and glycosylation of yeast invertase.